1. Field of the Invention
This invention pertains to the production and recovery of methyltransferase enzymes from bacteria containing cloned genes expressing the same. More specifically, a method is provided for producing and detecting bacterial having cloned genes coding for and expressing methylase (methyltransferase) enzymes, cultivating those bacteria, and recovering the expressed product, without prior knowledge of the nature of the gene sequence encoding said methylase enzyme.
2. Background of the Prior Art
The increasing importance of DNA sequencing, particularly, large fragment production for the construction of artificial genes and the like, has increased the importance of methylase or methyltransferase enzymes. DNA methylation is a basic post-replication event that protects DNA (particularly endogenous DNA) from restriction enzymes. Additionally, genes encoding methylase enzymes are frequently associated with restriction enzymes, which recognize the same sequence that the methyltransferase recognizes. Both restriction enzymes and the enzymes responsible for methylation are critical tools of recombinant DNA technology.
However, commercial use of the methyltransferase enzymes, particularly known DNA sequence specific methyltransferases has been limited by the low levels associated with the expression of the enzymes, in the natural expression vehicles, which include a wide variety of bacteria. Desirably, the commercial availability and utility of these enzymes can be increased by cloning of the gene sequence encoding for DNA specific methyltransferases in expression vehicles, together with an appropriate promoter sequence, if necessary, and over expressing them in bacteria such as E. coli.
However, hybrid plasmids encoding methyltransferases are restricted by the presence of one of a triplet of restriction enzymes common to E. coli strains, encoded by the genes mcrA, mcrB, or mrr (Raleigh et al), Proceedings of the National Academy of Science, U.S.A., 83 (1986). Specifically, it is believed that the presence of the restriction enzymes encoded by these genes are responsible for the degradation of the non-endogenous, "incoming" methylated plasmid DNA. Heitman et al, Journal of Bacteriology, 103 ( 1987 ).
Moreover, conventional gene cloning of DNA methylases depends on prior knowledge of the existence of the enzyme itself, and some information regarding the enzyme, or the gene responsible. Thus, in general, the cloning process begins with an identification of the DNA fragments encoding the enzyme or other protein in question. A DNA library is generated by use of restriction enzymes, dividing the original genome of the natural expression vehicle for the desired product at low levels into a plurality of gene fragments of different sizes.
These gene fragments are ligated into a cloning vector, and then taken up, by competent cells prepared according to conventional technology. The transformed cells are grown and tested for the presence of the desired activity.
As noted, a specific drawback of this process is that only predicted proteins, such as enzymes as to which there is considerable knowledge, can be obtained in this fashion. It is impossible, according to prior art processes, to rapidly identify unknown expressed DNA methylases in cloned hybrids, without exhaustive testing.
Accordingly, it remains an object of the art to provide a method whereby methyltransferases, both known and unstudied, can be successfully cloned into expression vehicles such as E. coli, and the methyltransferase expressed thereby recovered.